A major obstacle in ex vivo gene transfer has been the loss of transgene expression soon after implantation of the grafted transduced cells. Recently, a lentiviral vector system has been developed which has proven to express high levels of transgenes in vivo after direct injection into the tissue. In this study, we have investigated the use of such a vector for ex vivo gene transfer to the brain. A number of neural cell types were found to be permissive to transduction by the lentiviral vector in vitro and a majority of them expressed the transgene after transplantation to the rat brain. Transgene expression was detected up to 8 weeks post-grafting. These findings suggest that recombinant lentiviral vectors may be used for further development of ex vivo gene therapy protocols to the CNS.

Here we examined the ability of human neural progenitors from the embryonic forebrain, expanded for up to a year in culture in the presence of growth factors, to respond to environmental signals provided by the developing rat brain. After survival times of up to more than a year after transplantation into the striatum, the hippocampus, and the subventricular zone, the cells were analyzed using human-specific antisera and the reporter gene green fluorescent protein (GFP). From grafts implanted in the striatum, the cells migrated extensively, especially within white matter structures. Neuronal differentiation was most pronounced at the striatal graft core, with axonal projections extending caudally along the internal capsule into mesencephalon. In the hippocampus, cells migrated throughout the entire hippocampal formation and into adjacent white matter tracts, with differentiation into neurons both in the dentate gyrus and in the CA1–3 regions. Directed migration along the rostral migratory stream to the olfactory bulb and differentiation into granule cells were observed after implantation into the subventricular zone. Glial differentiation occurred at all three graft sites, predominantly at the injection sites, but also among the migrating cells. A lentiviral vector was used to transduce the cells with the GFP gene prior to grafting. The reporter gene was expressed for at least 15 weeks and the distribution of the gene product throughout the entire cytoplasmic compartment of the expressing cells allowed for a detailed morphological analysis of a portion of the grafted cells. The extensive integration and differentiation of in vitro-expanded human neural progenitor cells indicate that multipotent progenitors are capable of responding in a regionally specific manner to cues present in the developing rat brain.

We have examined long-term growth-factor expanded human neural progenitors following transplantation into the adult rat brain. Cells, obtained from the forebrain of a 9-week old fetus, propagated in the presence of epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor were transplanted into the striatum, subventricular zone (SVZ), and hippocampus. At 14 weeks, implanted cells were identified using antisera recognizing human nuclei and the reporter gene green fluorescent protein. Different migration patterns of the grafted cells were observed: (i) target-directed migration of doublecortin (DCX, a marker for migrating neuroblasts)-positive cells along the rostral migratory stream to the olfactory bulb and into the granular cell layer following transplantation into the SVZ and hippocampus, respectively; (ii) non-directed migration of DCX-positive cells in the grey matter in striatum and hippocampus, and (iii) extensive migration of above all nestin-positive/DCX-negative cells within white matter tracts. At the striatal implantation site, neuronal differentiation was most pronounced at the graft core with axonal projections extending along the internal capsule bundles. In the hippocampus, cells differentiated primarily into interneurons both in the dentate gyrus and in the CA1-3 regions as well as into granule-like neurons. In the striatum and hippocampus, a significant proportion of the grafted cells differentiated into glial cells, some with long processes extending along white matter tracts. Although the survival time was over 3 months in the present study a large fraction of the grafted cells remained undifferentiated in a stem or progenitor cell stage as revealed by the expression of nestin and/or GFAP.

The rat neural cell line RN33B has a remarkable ability to undergo region-specific neuronal differentiation after transplantation into the CNS. To further study its neurogenic properties in vivo, we used a recombinant lentiviral vector to genetically label the cells with the Green Fluorescent Protein (GFP) gene before implantation into the striatum/cortex, hippocampus, or mesencephalon of newborn rats. Three weeks after implantation, about 1-2% of the GFP-expressing cells had developed morphologies typical of neurons, astrocytes, or oligodendrocytes, the rest remained as either immature or undifferentiated nestin-positive cells. At 15-17 weeks postgrafting, the immature cells had disappeared in most graft recipients and only cells with neuronal or glial morphologies remained in similar numbers as at 3 weeks. The GFP distributed throughout the expressing cells, revealing fine morphological details, including dendrites with spines and extensive axonal projections. In all forebrain regions, the grafted cells differentiated into neurons with morphologies characteristic for each site, including large numbers of pyramidal-like cells in the cortex and the hippocampus, giving rise to dense projections to normal cortical target regions and to the contralateral hippocampus, respectively. In lower numbers, it was also possible to identify GFP-positive granulelike cells in the hippocampus, as well as densely spiny neurons in the striatum. In the mesencephalon by contrast, cells with astrocytic features predominated. The ability of the grafted RN33B cells to undergo region-specific differentiation into highly specialized types of forebrain projection neurons and establish connections with appropriate targets suggests that cues present in the microenvironment of the neonatal rat brain can effectively guide the development of immature progenitors, also in the absence of ongoing neurogenesis.